Phosphorus-containing dendrimers as transfection agents

ABSTRACT

The invention concerns phosphorus-containing dendrimers and their uses, as gene transfection agents, in vitro and in vivo, including in the treatment of human and animal diseases. These agents or vectors are in particular suited for delivering to appropriate target cells, nucleic acid sequences of interest.

The present invention relates to phosphorus-containing dendrimers and touses thereof, as agents for transfecting genes, in vitro and in vivo,including the treatment of human and animal diseases. Said agents orvectors are in particular suitable for delivering nucleic acid sequencesof interest to appropriate target cells.

Gene therapy is based on the therapeutic administration of nucleicacids; it requires the use of efficient and safe vectors for thetransfer of therapeutic genes and the success thereof therefore dependson the efficiency of gene transfer into the desired cells.

Many compositions capable of transfecting eukaryotic cells with aselective genetic material have already been described and essentiallybelong to two major types of transfection vectors:

-   -   viral transfection vectors, which are efficient but which have        limits for use: not tissue specific, necessity of obtaining        constructs for each gene of interest, potential risks for the        environment which lead to the setting up of expensive and        restrictive clinical infrastructures for the patient and the        personnel, and problems of immune response, of production of        viral particles by homologous recombination and of potential        oncogenic effects;    -   nonviral agents (synthetic vectors), capable of promoting the        transfer and expression of chemical substances, such as DNA, in        eukaryotic cells.

These synthetic vectors must essentially have two functions: condensethe DNA to be transfected and promote the cellular attachment thereofand also the passage thereof across the plasma membrane and, optionally,the nuclear membranes; in order to be efficient, such vectors musttherefore mimic the function of viruses. However, it appears that thevarious vectors provided in the prior art do not exhibit these twofunctions optimally and may also, depending on the cases, be toxic forthe cells.

Among these nonviral vectors, mention may be made in particular ofpreparations of cation lipids, which are particularly cytotoxic (15–18),techniques for encapsulating DNA in liposomes (9–11) and polycationicpolymers, such as poly(L-lysine), protamine, polyethyleneimine (12) orcationic dendrimers (polyamidoamines) (13, 14, 27), which associate withthe DNA via multiple electrostatic interactions engendering a process ofcooperativity, which produces particles called polyplexes, each onegiving variable transfection efficiencies depending on their structureand the cell type (25).

More precisely, dendrimers of the polyamidoamine (PAMAM) type arepolymers with a spherical and branched structure; they are soluble inaqueous solution and have a layer of primary amines at their surface;they are isomolecular and highly charged at their surface. The variousdendrimers described have various types of core: ammonium orethylenediamine (EDA), from which the polymerization process isinitiated. These PAMAM dendrimers contain a defined number of aminogroups at the surface of the polymer, which are positively charged atphysiological pH. It has been shown that such molecules interact withanions such as nucleic acids and that the DNA/dendrimer complexes arecapable of transfecting cells in a way similar to that observed withDNA/polylysine complexes, but with greater efficiency, linked to theirsolubility and to their structure. In particular, G₃–G₅ PAMAMdendrimers, comprising, as the core, either NH₃ groups or EDA groups,are capable of forming stable complexes with DNA under physiologicalconditions, and G₅–G₁₀ dendrimers are capable of transfecting genes. Thefact that the capacity of transfection is limited to this group ofdendrimers is doubtless linked to the number of amino groups present attheir surface and to their spherical shape. The efficiency of the latterdepends on the chemical modifications to the initial dendrimericstructure by heat treatment which results in a population ofheterodispersed compounds (15, 21).

Over about ten years, research on the chemistry of dendrimers hasdeveloped considerably, due to their structure (ordered polyfunctionalpolymers) and to their particular properties linked to the presence of aconsiderable number of functionalities at their ends, which provide alarge number of surfaces and interfaces, and to the presence of emptyzones which allow encapsulation of diverse molecules; this research hasled to the synthesis of other structures corresponding tophosphorus-containing dendrimers, such as those described by C. Galliotet al. (18), by C. Larré et al. (19) or by D. Prévôté et al. (28).

However, it emerges from the literature that these phosphorus-containingdendrimers, which are not water-soluble, cannot be used as transfectionagents.

In the context of their research, the inventors have now shown thatnovel phosphorus-containing dendrimers which are functionalized at thelevel of the inner layers and which comprise protonated tertiary aminesas ends, have particularly advantageous properties as vectors fortransferring nucleic acids.

A subject of the present invention is polycationic phosphorus-containingdendrimers, characterized in that they consist:

-   -   of a central layer in the form of a core P₀ comprising 2 to 8        functionalized groups and, in particular, the group of general        formula Ia (also named N₃P₃):    -   or the group of general formula Ib:        -   of n intermediate layers, which may be identical or            different, each one of said intermediate layers consisting            of units P1 corresponding to formula II:    -   in which:    -   L is an oxygen, phosphorus or sulfur atom,    -   M represents one of the following groups:        -   an aromatic group di-, tri- or tetra-substituted with alkyl            groups, alkoxy groups or unsaturated groups of the C₁–C₁₂            olefinic, azoic or acetylenic type, all these groups            possibly incorporating phosphorus, oxygen, nitrogen, sulfur            or halogen atoms, or        -   an alkyl or alkoxy group comprising several substituents as            defined when M is an aromatic group,    -   R₁ and R₂, which may be identical or different, represent a        hydrogen atom or one of the following groups: alkyl, alkoxy,        aryl, possibly comprising phosphorus, oxygen, sulfur, nitrogen        or halogen atoms, with R₂ most commonly being different from R₁,    -   n is an integer between 1 and 11,    -   E is an oxygen, sulfur or nitrogen atom, said nitrogen atom        possibly being linked to an alkyl, alkoxy or aryl group, all        these groups possibly incorporating phosphorus, oxygen,        nitrogen, sulfur or halogen atoms,        -   an outer layer consisting of units P2, which may be            identical or different, corresponding to formula III:    -   in which:    -   R₅ represents a hydrogen atom or one of the following groups:        alkyl, alkoxy, aryl, these groups possibly comprising        phosphorus, oxygen, nitrogen, sulfur or halogen atoms,    -   W represents one of the following groups: alkyl, alkoxy, aryl,        all these groups possibly comprising phosphorus, oxygen,        nitrogen, sulfur or halogen atoms,    -   R₃ and R₄, which may be identical or different, represent a        C₁–C₅ alkyl group,    -   X represents a hydrogen atom or a C₁–C₅ alkyl group or is        absent, and    -   Z represents a halide ion, an alkylCOO⁻ group or any other        anionic group comprising carbon, oxygen, sulfur, nitrogen,        phosphorus or halogen atoms, or is absent.

A preferred group of formula II is in particular the following group:

-   -   a preferred group of formula III is in particular the following        group:    -   in which p=1 to 5

Such compounds represent dendrimers and are in particular represented inFIGS. 1 to 3.

The expression “alkoxy” denotes the radicals of general formula R′O—,for example methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy,tert-butoxy groups.

The expression “alkyl” denotes the linear- or branched-chain radicalscontaining up to 8 carbon atoms. Among these radicals, mention may bemade of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,tert-butyl, pentyl, hexyl, heptyl or octyl radicals.

The expression “aryl” denotes, for example, a phenyl radical optionallysubstituted with one or more alkoyl or alkoxyl radicals or with achlorine, bromine or fluorine atom, or a 5- or 6-membered aromaticheterocyclic radical containing 1 to 2 hetero atoms such as nitrogen oroxygen. Among these aryl radicals, mention may be made of (o-, m-, orp-)phenyl, (3,4-, 2,6-, 2,3-)methoxyphenyl, (o-, m-, orp-)dimethoxyphenyl, tolyl, thienyl or pyridyl radicals.

It is possible to obtain several types of product designated, ingeneral, by the formula A—[G_(n)], in which A defines the type ofterminal unit and G_(n) defines the number of layers of intermediateunits P1 (corresponding to the number of generations):

-   -   the series of compounds 2-[G_(n)] has terminal units of the said        formula III, in which X represents a hydrogen atom, p is equal        to 2, R₃ and R₄ are identical and represent ethyl groups, Z is a        chloride ion and n is an integer between 1 and 11, preferably        between 1 and 10 (see FIG. 3);    -   the series of compounds 3-[G_(n)] has terminal units of formula        III, in which X is empty, p is equal to 2, R₃ and R₄ are        identical and represent ethyl groups, Z is empty and n is an        integer between 1 and 11, preferably between 1 and 10 (see FIG.        3);    -   the series of compounds 4-[G_(n)] has terminal units of formula        III, in which X represents a methyl group, p is equal to 2, R₃        and R₄ are identical and represent ethyl groups, Z is an iodide        ion and n is an integer between 1 and 11, preferably between 1        and 10 (see FIG. 3);    -   the series of compounds 5-[G_(n)] has terminal units of formula        III, in which X represents a methyl group, p is equal to 2, R₃        and R₄ are identical and represent ethyl groups, Z is a group        CH₃COO⁻ and n is an integer between 1 and 11, preferably between        1 and 10 (see FIG. 3);    -   the series of compounds 1-[G_(n)] has Cl₂ terminal units and n        is an integer between 1 and 11, preferably between 1 and 10 (see        FIG. 3) (intermediate products for the preparation of the        dendrimers according to the invention).

The polycationic phosphorus-containing dendrimers according to theinvention have a certain number of advantages compared to the dendrimersof the prior art used as gene vectors:

-   -   they are isomolecular and, as a result, are reproducible (high        degree of purity),    -   they are charged, which confers on them a particular affinity        with respect to the nucleic acids to be transferred and        therefore good transporter quality,    -   they can be functionalized both at the surface and in the inner        layers,    -   they are water-soluble without degradation within a large pH        range (3 to 12) (stability in aqueous solution for several        months). This represents a clear advantage compared with the        dendrimers of the PAMAM type which must be heat-degraded        (relatively poorly reproducible step) to give compounds which        are active in transfection,    -   they are relatively noncytotoxic (viability of transfected cells        greater than 80%).

The phosphorus-containing dendrimers according to the invention may beobtained, in a reproducible manner, by controlled growth of thedendrimeric structure by addition of successive layers of motifs orunits H₂N—N(R₂)—P(S)Cl₂ of formula VIII (example of product of formulaII). The central core or block of formula VII (see FIGS. 1 and 2)correspond to a hexachlorocyclotriphosphazene unit (Cl₆N₃P₃) modified bythe triethylammonium of 4-hydroxybenzaldehyde. This core, whichcomprises 6 terminal aldehyde functions, is brought into contact with adichlorophosphonoalkylhydrazide of formula VIII H₂N—N(Alk)-P(S)Cl₂ toproduce a dendron comprising dichlorothiophosphine, P(S)Cl₂, terminalunits, which can be brought into contact once more with a4-hydroxybenzaldehyde salt of formula VI, to produce, by iteration, eachdendrimer generation. The 1st, 2nd, 3rd, 4th and 5th dendrimergenerations comprise, respectively, 6, 12, 24, 48 and 96 Cl₂ terminalunits, which can be treated with N,N-dialkylethylenediamine, to producecationic dendrimers after protonation: 2-[G₁] to 2-[G₅], in accordancewith FIG. 3.

The polycationic phosphorus-containing dendrimers have 12, 24, 48, 96 or192 peripheral positive charges, respectively, for the 1st, 2nd, 3rd,4th and 5th dendrimer generations.

The methylated forms (5-[G₁] to 5-[G₅]) are prepared from thecorresponding neutral terminal amines (3-[G₁] to 3-[G₅]) by methylationin the presence of methyl iodide, followed by an iodide/acetate exchangeusing a suitable resin (see example 1).

The purity and integrity of the dendrimers is verified by spectralanalysis (¹H, ¹³C and ³¹P NMR).

Only 8 to 10% of the terminal branches of the methylated dendrimers(5-[G₁] to 5-[G₅]) exhibit a deficit in methyl groups.

More precisely, the dendrimers according to the invention may beprepared in the following way:

-   -   (1) reaction of a product of formula V (also named N₃P₃)    -   in which R₁ represents a group comprising an aldehyde function        of formula VI:    -   so as to obtain a product of formula VII: N₃P₃(OC₆H₄CHO)₆        comprising 6 aldehyde functions (central layer consisting of a        core P₀; FIG. 3),    -   (2) reaction of the product of formula VII obtained in (1) with        a dichlorophosphonohydrazide of formula VIII: H₂N—N(R₂)—P(S)Cl₂        to produce a dendron of the 1-[G_(n)] type comprises Cl₂        terminal units,    -   (3) reiteration of steps (1) and (2) on the product obtained        in (2) to produce a number n of intermediate layers; the 1st,        2nd, 3rd, 4th and 5th dendrimer generations comprise,        respectively, 6, 12, 24, 48 and 96 Cl₂ terminal units, and    -   (4) treatment of said Cl₂ terminal units with        N,N-dialkylethylenediamine, to produce cationic dendrimers        according to the invention after protonation.

A subject of the present invention is also a composition capable ofacting as an agent for transfecting a nucleic acid sequence into aeukaryotic cell, characterized in that it comprises a nucleic acid and apolycationic phosphorus-containing dendrimer as defined above, coupledto said nucleic acid.

According to an advantageous embodiment of said composition, it alsocomprises at least one pharmaceutically acceptable vehicle.

According to another advantageous embodiment of said composition, theN/P ratio, in which N corresponds to the terminal cationic groups of thedendrimer (charged amines) and P corresponds to the phosphate groups ofsaid nucleic acid, is between 5 and 10.

According to another advantageous embodiment of said composition, italso comprises an agent for permeabilizing the membrane, capable oftransporting said nucleic acid across the cytoplasmic or endosomalmembranes of said eukaryotic cell.

According to yet another advantageous embodiment of said composition,said polycationic phosphorus-containing dendrimer is associatednoncovalently with said nucleic acid.

Advantageously, the polycationic phosphorus-containing dendrimersaccording to the invention, selected in the series 2-[G_(n)] in whichn=3–5, are particularly advantageous as vectors for transferring nucleicacid, while the dendrimers of the series 5-[G_(n)] are toxic andrelatively inefficient in transfecting nucleic acids into eukaryoticcells, both in the presence and absence of serum.

This phenomenon is perhaps linked to a high density of stable positivecharges, which may cause rupturing of the cell membrane and thus lead tocell death. It is not possible to decrease the charge density for thealkylated products without degrading the dendrimers, whereas the chargedensity of the series 2-[G_(n)] may be modulated by microenvironmentalmodifications of the pH at the level of the cell membrane. In addition,the possibility of modulating the charge density of this series ofdendrimers may constitute a key factor in the release of the genetransported in the endosomes. These dendrimers thus perhaps act as aproton reservoir in the cellullar compartments, their charge densitybeing controlled by ATPase-dependent proton pumps and by modificationsof the intracellular chloride concentration. The possibility ofdecreasing the cationic charge density of these dendrimers outside andinside the cells should be favorable to their use in vivo.

Besides the arrangements above, the invention also comprises otherarrangements, which will emerge from the following description, whichrefers to examples of implementation of the method which is the subjectof the present invention, and also to the attached drawings in which:

FIG. 1 illustrates the structure of the central core P₀ (N₃P₃) and thedendrimer according to the invention of the 3-[G₂] type;

FIG. 2 illustrates a dendrimer according to the invention of the 2-[G₄]type;

FIG. 3 illustrates a method for preparing the dendrimers according tothe invention;

FIG. 4 illustrates the gene transfer properties of the dendrimers of the2-[G_(n)] type, in the presence of serum (right-hand sections of FIGS.4A and 4B) or in the absence of serum (left-hand sections of FIGS. 4Aand 4B);

FIG. 5 illustrates the gene transfer properties of the dendrimers of the5-[G_(n)] type, in the presence of serum (right-hand sections of FIGS.5A and 5B) or in the absence of serum (left-hand sections of FIGS. 5Aand 5B).

It should be clearly understood, however, that these examples are givenonly by way of illustration of the subject of the invention, in whichthey in no way constitute a limitation.

EXAMPLE 1 Preparation of the Dendrimers According to the Invention

General Methods and Products Used

All the manipulations were carried out using common techniques underconditions of a strong vacuum or under argon. The ¹H, ³¹P and ¹³C NMRspectra were recorded on Brucker spectrometers (AC80, AC200 and AC250).The ³¹P NMR chemical shifts of the reagents are expressed in ppmrelative to 85% of H₃PO₄. The numbering used for the NMR is detailed onFIG. 1. The 1-[G_(n)] dendrimers were synthesized according to publishedprotocols (16, 17). In the abbreviation 1-[G_(n)], the number 1corresponds to a dendrimer with ends C1, 2, 3, 4 or 5 for ends —NH(Et)₂⁺Cl⁻, —N(Et)₂, —NMe(Et)₂ ⁺I⁻ or —NMe(Et)₂ ⁺OAc—, respectively, and ncorresponds to the number of generations of the dendrimer (number ofintermediate layers). The methyl iodide and theN,N-diethylethylenediamine were supplied by Aldrich and the AG1-X8resin, which has a high capacity for anion exchange, was supplied byBiorad.

General Procedures for Synthesizing the Products of the 2-[G_(n)] Type

N,N-Diethylethylenediamine (n=1, 93 μl, 0.66 mmol; n=2, 71 μl, 0.5 mmol;n=3, 68 μl, 0.48 mmol; n=4, 61 μl, 0.43 mmol; n=5, 60 μl, 0.42 mmol) wasadded dropwise, using a syringe, to a solution containing 100 mg ofdendrimer 1-[G_(n)] (n=1, 55 μmol; n=2, 21 μmol; n=3, 10 μmol; n=4, 4.5μmol; n=5, 2.2 μmol) in 15 ml of distilled THF (THFΔ), with vigorousstirring. After stirring overnight at room temperature (RT), the solventwas removed by filtration. The white powder obtained was washed twicewith 20 ml of distilled THF and dried by evaporation. The protonsproduced during the coupling reaction were captured by the terminaltertiary amine residues and, consequently, the 2-[G_(n)] dendrimers wereobtained in the form of chlorides.

The first generation of dendrimer 2-[G₁] is obtained with a yield of80%. The NMR data are as follows:

³¹P {¹H} NMR (CD₃OD): δ=7.9 (P₀), 69.6 (P₁).

¹H NMR (d₆-DMSO): δ=1.3 (t, ³J_(HH)=6.3 Hz, 72H, CH₂CH₃), 3.0–3.5 (m,114H, CH₃—N—P₁, CH₂), 5.7 (br m, 12H, N—H), 7.1 (d, ³J_(HH)=8.4 Hz, 12H,C₀ ²—H), 7.9 (s, 6H, CH═N), 7.9, (d, ³J_(HH)=8.4 Hz, 12H, C₀ ³—H), 10.8(br s, 12H, ⁺N—H).

¹³C {¹H} NMR (CD₃OD): δ=9.7 (s, CH₂CH₃), 33.3 (d, ²J_(CP1)=10.3 Hz,CH₃—N—P₁), 37.9 (s, CH₂—N—P₁), 49.5 (s, CH₂CH₃), 53.9 (d, ³J_(CP1)=6.2HZ, CH₂—CH₂—N—P₁) 122.6 (s, C₀ ²), 129.8 (s, C₀ ³), 135.0 (s, C₀ ⁴),139.3 (d, ³J_(CP1)=11.6 Hz, CH═N), 152.4 (d, ²J_(CP0)=7.3 Hz, C₀ ¹).

UV-vis (H₂O): λ_(max)(ε, M⁻¹×cm ⁻¹) 284 nm (1.2×10⁵).

Second generation of dendrimer 2-[G₂] (yield=95%):

³¹P {¹H} NMR (CD₃OD): δ=8.5 (P₀), 62.0 (P₁), 69.6 (P₂). ¹H NMR(d₆-DMSO): δ=1.3 (br s, 144H, CH₂CH₃), 3.0–3.6 (br m, 246H,CH₃—N—P_(1,2), CH₂), 5.6 (br m, 24H, N—H), 7.0–7.4 (br m, 36H, C₀ ²—H,C₁ ¹—H), 7.7–8.2 (m, 54H, CH═N, C₀ ³—H, C₁ ³—H), 10.7 (br s, 24H, ⁺N—H).

¹³C {¹H} NMR (CD₃OD): δ=9.6 (s, CH₂CH₃), 33.0 (d, ²J_(CP2)=10.6 Hz,CH₃—N—P₂), 34.2 (d, ²J_(CP1)=11.8 Hz, CH₃—N—P₁), 37.8 (s, CH₂—N—P₂),49.2 (s, CH₂CH₃), 53.9 (d, ³J_(CP2)=6.3 Hz, CH₂—CH₂—N—P₂), 122.8 (s, C₀²), 123.0 (d, ³J_(CP1)=3.0 Hz, C₁ ²), 129.7 (s, C₁ ³), 130.0 (S, C₀ ³),134.3 (s, C₀ ⁴), 135.0 (s, C₁ ⁴), 139.1 (d, ³J_(CP2)=12.5 Hz, CH═N),141.3 (d, ³J_(CP1)=15.4 Hz, CH═N), 152.6 (d, ²J_(CP1)=7.3 Hz, C₁ ¹),152.6 (s, C₀ ¹).

UV-vis (H₂O): λ_(max)(ε, M⁻¹×cm⁻¹) 284 nm (3.1×10⁵).

Third generation of dendrimer 2-[G₃] (yield=95%):

³¹P {¹H} NMR (CD₃OD): δ=8.6 (P₀), 61.5 (P₁), 62.3 (P₂), 69.5 (P₃).

¹HNMR (d₆-DMSO): δ=1.3 (br s, 288H, CH₂CH₃), 3.0–3.5 (br m, 510H,CH₃—N—P_(1,2,3), CH₂), 5.7 (br s, 48H, N—H), 7.0–7.5 (br m, 84H, C₀ ²—H,C₁ ²—H, C₂ ²—H), 7.7–8.2 (br m, 126H, CH═N, C₀ ³—H, C₁ ³—H, C₂ ³—H),10.8 (br s, 48H, ⁺N—H).

¹³C {¹H} NMR (CD₃OD): δ=9.6 (s, CH₂CH₃), 33.1 (d, J_(CP3)=9.4 Hz,CH₃—N—P₃), 34.2 (m, CH₃—N—P_(1,2)), 37.6 (s, CH₂—N—P₃), 49.2 (s,CH₂CH₃), 53.7 (d, ³J_(CP3)=6.3 Hz, CH₂—CH₂—N—P₃), 123.2 (br s, C₀ ², C₁², C₂ ²), 129.6 (br s, C₀ ³, C₁ ³, C₂ ³), 134.0 (s, C₀ ⁴, C₁ ⁴), 134.8(s, C₂ ⁴), 139.0 (br s, C₂—CH═N), 141.4 (br s, CH═N), 152.4 (d,J_(CP2)=7.3 Hz, C₂ ¹), 152.8 (br s, C₀ ¹, C₁ ¹).

UV-vis (H₂O): λ_(max)(ε, M⁻¹×cm⁻¹) 286 nm (7.3×10⁵).

Fourth generation of dendrimer 2-[G₄] (yield=95%):

³¹P {¹H} NMR (CD₃OD): δ=8.4 (P₀), 62.0 (P_(1,2,3)), 69.4 (P₄).

¹H NMR (d₆-DMSO): δ=1.3 (br s, 576H, CH₂CH₃), 3.0–3.5 (m, 1038H,CH₃—N—P_(1,2,3,4), CH₂), 5.6 (br s, 96H, N—H), 7.0–7.5 (br m, 180H, C₀²—H, C₁ ²—H, C₂ ²—H, C₃ ²—H), 7.7–8.2 (m, 270H, CH═N, C₀ ³—H, C₁ ³—H, C₂³—H, C₃ ³—H), 10.8 (br s, 96H, ⁺N—H).

¹³C {¹H} NMR (CD₃OD): δ=9.7 (s, CH₂CH₃), 33.2 (d, ²J_(CP4)=9.2 Hz,CH₃—N—P₄), 34.3 (d, ²J_(CP)=10.1 Hz, CH₃—N—P_(1,2,3)), 37.7 (s,CH₂—N—P₄), 49.2 (s, CH₂CH₃), 53.8 (d, ³J_(CP4)=5.5 Hz, CH₂—CH₂—N—P₄),123.1 (br s, C₀ ², C₁ ², C₂ ², C₃ ²) 129.7 (br s, C₀ ³, C₁ ³, C₂ ³, C₃³), 134.2 (s, C₀ ⁴, C₁ ⁴, C₂ ⁴), 134.9 (s, C₃ ⁴), 139.2 (br s, C₃⁴—CH═N), 141.5 (br s, CH═N), 152.5 (d, ³J_(CP3)=7.4 Hz, C₃ ¹), 153.0 (brs, C₀ ¹, C₁ ¹, C₂ ¹).

UV-vis (H₂O): λ_(max) (ε, M⁻¹×cm⁻¹) 288 nm (1.7×10⁶).

Fifth generation of dendrimer 2-[G₅] (yield=95%):

³¹P {¹H} NMR (CD₃OD): δ=62.0 (P_(1,2,3,4)), 69.3 (P₅).

¹H NMR (d₆-DMSO): δ=1.3 (br s, 1152H, CH₂CH₃), 2.9–3.5 (br m, 2094H,CH₃—N—P_(1,2,3,4,5), CH₂), 5.6 (br s, 192H, N—H), 7.0–7.5 (m, 372H, C₀²—H, C1²—H, C₂ ²—H, C₃ ²—H, C₄ ²—H), 7.7–8.2 (m, 558H, CH═N, C₀ ³—H, C₁³—H, C₂ ³—H, C₃ ³—H, C₄ ³—H), 10.8 (br s, 192H, N—H).

¹³C {¹H} NMR (CD₃OD): δ=9.7 (s, CH₂CH₃), 33.2 (br s, CH₃—N—P₅), 34.3 (brs, CH₃—N—P_(1,2,3,4)), 37.8 (s, CH₂—N—P₅), 49.2 (s, CH₂CH₃), 53.8 (s,CH₂—CH₂—N—P₅), 123.1 (br s, C₀ ², C₁ ², C₂ ², C₃ ², C₄ ²), 129.7 (br s,C₀ ³, C₁ ³, C₂ ³, C₃ ³, C₄ ³), 134.2 (s, C₀ ⁴, C₁ ⁴, C₂ ⁴, C₃ ⁴), 134.9(s, C₄ ⁴), 139.2 (br s, C₄ ⁴—CH═N), 141.5 (br s, CH═N), 152.5 (s, C₄ ¹),153.0 (br s, C₀ ¹, C₁ ¹, C₂ ¹, C₃ ¹).

UV-vis (H₂O): λ_(max) (ε, M⁻¹×cm⁻¹) 286 nm (3.3×10⁶).

General Procedures for Synthesizing the Products of the 3-[G_(n)] Type

A normal sodium hydroxide solution (n=1, 372 ml, 0.37 mmol; n=2, 312 ml,0.31 mmol; n=3, 288 ml, 0.29 mmol; n=4, 288 ml, 0.28 mmol; n=5, 288 ml,0.28 mmol) was added dropwise to a solution containing 100 mg ofdendrimer 2-[G_(n)] in 30 ml of distilled water (n=1, 31 mmol; n=2, 13mmol; n=3, 6 mmol; n=4, 3 mmol; n=5, 1.5 mmol), with vigorous stirring.The precipitate was isolated by centrifugation and dissolved inchloroform, and the organic layer was then dried on sodium sulfate.Finally, the product is filtered and dried by evaporation.

Dendrimer 3-[G₁] (yield=80%):

³¹P {¹H} NMR (CDCl₃): δ=8.2 (P₀), 68.3 (P₁).

¹H NMR (CD₂Cl₂): δ=0.9 (t, ³J_(HP1)=7.0 Hz, 72H, CH₂CH₃), 2.3–2.5 (m,72H, CH₂—N(CH₂—CH₃)₂), 2.9 (m, 24H, CH₂—N—P₁), 3.1 (d, ³J_(HP1)=9.4 Hz,18H, CH₃N—P₁), 4.0 (m, 12H, N—H), 6.9 (d, ³J_(HH)=8.5 Hz, 12H, C₀ ²—H),7.5 (s, 6H, CH═N), 7.5 (d, ³J_(HH)=8.5 Hz, 12H, C₀ ³—H).

¹³C {¹H} NMR (CDCl₃): δ=11.4 (s, CH₂CH₃), 30.5 (d, ²J_(CP1)=10.7 Hz,CH₃—N—P₁), 38.4 (s, CH₂—N—P₁), 46.3 (s, CH₂CH₃), 52.9 (d, ³J_(CP1)=7.8Hz, CH₂—CH₂—N—P₁), 120.8 (s, C₀ ²), 127.3 (s, C₀ ³), 132.7 (s, C₀ ⁴)135.4 (d, ³J_(CP1)=12.5 Hz, CH═N), 150.4 (d, ²J_(CP0)=5.1 Hz, C₀ ¹).

Dendrimer 3-[G₂] (yield=95%):

³¹P {¹H} NMR (CDCl₃): δ=8.4 (P₀), 62.9 (P₁), 68.1 (P₂). ¹H NMR (CD₂Cl₂):δ=0.9 (t, ³J_(HH)=7 Hz, 144H, CH₂CH₃), 2.2–2.5 (m, 144H,CH₂—N(CH₂—CH₃)₂), 2.9 (m, 48H, CH₂—N—P₂), 3.0 (d, ³J_(HP2)=9.2 Hz, 36H,CH₃N—P₂), 3.2 (d, ³J_(HP1)=10 Hz, 18H, CH₃—N—P₁), 4.0 (br m, 24H, N—H),6.9–7.1 (m, 36H, C₀ ²—H, C₁ ²—H), 7.4–7.7 (m, 54H, CH═N, C₀ ³—H, C₁³—H).

¹³C {¹H} NMR (CDCl₃): δ=11.4 (s, CH₂CH₃), 30.6 (d, ²J_(CP2)=10.8 Hz,CH₃—N—P₂), 32.9 (d, ²J_(CP1)=11.8 Hz, CH₃—N—P₁), 38.3 (s, CH₂—N—P₂),46.3 (s, CH₂CH₃), 52.9 (d, ³J_(CP2)=7.8 Hz, CH₂—CH₂—N—P₂), 121.1 (s, C₀², C₁ ²), 127.4 (s, C₁ ³), 128.1 (s, C₀ ³), 132.0 (s, C₀ ⁴), 133.1 (S,C₁ ⁴), 135.1 (d, ³J_(CP2)=12.4 Hz, CH═N), 138.6 (d, ³J_(CP1)=15.4 Hz,CH═N), 150.3 (d, ²J_(CP1)=7.3 Hz, C₁ ¹), 151.1 (s, C₀ ¹).

Dendrimer 3-[G₃] (yield=95%):

³¹P {¹H} NMR (CDCl₃): δ=8.5 (P₀), 62.9 (P_(1,2)), 68.1 (P₃).

¹H NMR (CD₂Cl₂): δ=0.9 (t, ³J_(HH)=6.4 Hz, 288H, CH₂CH₃), 2.2–2.5 (br m,288H, CH₂—N(CH₂CH₃)₂), 2.9 (br m, 96H, CH₂—N—P₃), 3.0 (d, ³J_(HP3)=9.2Hz, 72H, CH₃—N—P₃), 3.2–3.4 (br m, 54H, CH₃—N—P_(1,2)), 4.0 (br s, 48H,N—H), 6.9–7.3 (br m, 84H, C₀ ²—H, C₁ ²—H, C₂ ²—H, 7.4–7.7 (br m, 126H,CH═N, C₀ ³—H, C₁ ³—H, C₂ ³—H).

¹³C {¹H} NMR (CDCl₃): δ 11.4 (s, CH₂CH₃), 30.6 (d, ²J_(CP3)=10.3 Hz,CH₃—N—P₃), 32.5 (d, ²J_(CP)=12.7 Hz, CH₃—N—P_(1,2)), 38.4 (s, CH₂—N—P₃),46.4 (s, CH₂CH₃), 53.0 (d, ³J_(CP3)=7.9 Hz, CH₂—CH₂—N—P₃), 121.4 (s, C₀², C₁ ², C₂ ²), 127.5 (s, C₂ ³), 128.2 (s, C₀ ³, C₁ ³), 132.0 (s, C₀ ⁴),132.3 (s, C₁ ⁴), 133.1 (s, C₂ ⁴), 135.2 (d, ³J_(CP3)=12.1 Hz, CH═N),138.8 (d, ³J_(CP3)=12.1 Hz, CH═N), 150.4 (d, ²J_(CP3)=6.8 Hz, C₂ ¹),151.2 (d, ²J_(CP)=6.3 Hz, C₀ ¹, C₁ ¹).

Dendrimer 3-[G₄] (yield=95%):

³¹P {¹H} NMR (CDCl₃): δ=8.4 (P₀), 62.4 (P_(1,2,3)), 68.0 (P₄).

¹H NMR (CD₂Cl₂): δ=0.8 (br s, 576H, CH₂CH₃), 2.4 (br s, 576H,CH₂—N(CH₂CH₃)₂), 2.6–3.4 (br m, 462H, CH₃—N—P_(1,2,3,4), CH₂—N—P₄), 4.0(br m, 96H, N—H), 7.0–7.7 (m, 450H, C₆H₄, CH═N).

¹³C {¹H} NMR (CDCl₃): δ=11.6 (s, CH₂CH₃), 30.6 (d, ²J_(CP4)=10.4 Hz,CH₃—N—P₄), 33.0 (d, ²J_(CP)=12.6 Hz, CH₃—N—P_(1,2,3)), 38.5 (s,CH₂—N—P₄), 46.4 (s, CH₂CH₃), 53.1 (d, ³J_(CP4)=7.7 Hz, CH₂—CH₂—N—P₄),121.5 (s, C₃ ²), 121.8 (s, C₀ ², C₁ ², C₂ ²), 127.5 (s, C₃ ³), 128.3 (s,C₀ ³, C₁ ³, C₂ ³), 132.2 (s, C₀ ⁴, C₁ ⁴, C₂ ⁴), 133.2 (s, C₃ ⁴), 135.1(d, ³J_(CP4)=12.0 Hz, CH═N), 138.7 (br s, CH═N), 150.5 (d, ²J_(CP2)=7.4Hz, C₃ ¹), 151.3 (br m, C₀ ¹, C₁ ¹, C₂ ¹).

Dendrimer 3-[G₅] (yield=95%):

³¹P {¹H} NMR (CDCl₃): δ=62.4 (P_(1,2,3,4)), 68.0 (P₅).

¹H NMR (CD₂Cl₂): δ=1.0 (br s, 1152H, CH₂CH₃), 2.4 (br s, 1152H,CH₂—N(CH₂CH₃)₂), 2.7–3.5 (br m, 942H, CH₃—N—P_(1,2,3,4,5), CH₂—N—P₅),4.1 (m, 192H, N—H), 7.0–7.8 (m, 930H, C₆H₄, CH═N).

¹³C {¹H} NMR (CDCl₃): δ=11.5 (s, CH₂CH₃), 30.5 (d, ²J_(CP5)=11.1 Hz,CH₃—N—P₅), 33.0 (d, ²J_(CP)=12.3 Hz, CH₃—N—P_(1,2,3,4)), 38.4 (s,CH₂—N—P₅), 49.4 (s, CH₂CH₃), 53.0 (d, ³J_(CP5)=8.1 Hz, CH₂—CH₂—N—P₅),121.4 (s, C₄ ²), 121.7 (s, C₀ ², C₁ ², C₂ ², C₃ ²), 127.4 (s, C₄ ³),128.1 (s, C₀ ³, C₁ ³, C₂ ³, C₃ ³), 132.1 (s, C₀ ⁴, C₁ ⁴, C₂ ⁴, C₃ ⁴),133.1 (s, C₄ ⁴) 135.1 (d, ³J_(CP5)=12.1 Hz, CH═N), 138.7 (br s, CH═N),150.5 (d, ²J_(CP3)=6.6 Hz, C₄ ¹), 151.2 (d, ²J_(CP)=6.2 Hz, C₀ ¹, C₁ ¹,C₂ ¹, C₃ ¹).

General Procedures for Synthesizing the Products of the 4-[G_(n)] Type

A solution comprising 100 mg of neutral dendrimers 3-[G_(n)] (n=1, 36mmol; n=2, 15 mmol; n=3, 7 mmol; n=4, 3.3 mmol; n=5, 1.6 mmol) andmethyl iodide (n=1, 27 μl, 0.43 mmol; n=2, 22 μl, 0.36 mmol; n=3, 21 μl,0.34 mmol; n=4, 20 μl, 0.32 mmol; n=5, 19 μl, 0.31 mmol) in 15 ml of DMFwas stirred overnight at room temperature. The solution was dried byevaporation. The paste obtained was washed with 20 ml of a pentane/ether(1/1, v/v) mixture so as to obtain a yellow powder of methylateddendrimers named 4-[G_(n)].

Dendrimer 4-[G₁] (yield=90%):

³¹P {¹H} NMR (d₆-DMSO): δ=7.3 (P₀), 68.1 (P₁).

¹H NMR (d₆-DMSO): δ=1.3 (t, ³J_(HH)=6.4 Hz, 72H, CH₂CH₃), 3.1 (s, 36H,⁺N—CH₃), 3.2 (d, ³J_(HP1)=10.4 Hz, 18H, CH₃—N—P₁), 3.3–3.7 (br s, 96H,CH₂), 5.5 (br m, 12H, N—H), 7.1 (d, J_(HH)=8.1 Hz, 12H, C₀ ³—H), 8.0 (s,6H, CH═N), 8.0 (d, ³J_(HH)=8.1 Hz, 12H, C₀ ³—H).

¹³C {¹H} NMR (d₆-DMSO): δ=7.8 (s, CH₂CH₃), 32.6 (d, ²J_(CP1)=9.5 Hz,CH₃—N—P₁), 34.9 (s, CH₂—N—P₁), 47.3 (s, ⁺N—CH₃), 56.4 (s, CH₂CH₃), 58.8(s, CH₂—CH₂—N—P₁), 120.7 (s, C₀ ²), 128.3 (s, C₀ ³), 133.2 (s, C₀ ⁴),137.4 (d, ³J_(CP1)=14.2 Hz, CH═N), 149.9 (s, C₀ ¹).

Dendrimer 4-[G₂] (quantitative yield):

³¹P {¹H} NMR (d₆-DMSO): δ=7.3 (P₀), 61.7 (P₁), 68.1 (P₂).

¹H NMR (d₆-DMSO): δ=1.3 (t, ³J_(HH)=6.2 Hz, 144H, CH₂CH₃), 3.1 (s, 72H,⁺N—CH₃), 3.2–3.6 (m, 246H, CH₃—N—P_(1,2), CH₂), 5.7 (br s, 24H, N—H),7.0–7.4 (m, 36H, C₀ ²—H, C₁ ²—H), 7.7–8.2 (m, 54H, CH═N, C₀ ³—H, C₁³—H).

¹³C {¹H} NMR (d₆-DMSO): δ=7.8 (s, CH₂CH₃), 32.2 (d, ²J_(CP2)=8.9 Hz,CH₃—N—P₂), 33.5 (d, ²J_(CP1)=12.9 Hz, CH₃—N—P₁), 34.9 (s, CH₂—N—P₂),47.3 (s, +N—CH₃), 56.4 (s, CH₂CH₃), 58.8 (d, ³J_(CP2)=3.3 Hz,CH₂—CH₂—N—P₂), 121.3 (s, C₁ ², C₀ ²), 128.2 (s, C₁ ³), 128.6 (s, C₀ ³),132.3 (s, C₀ ⁴), 133.2 (s, C₁ ⁴), 137.1 (d, ³J_(CP2)=12.3 Hz, CH═N),140.7 (br s, CH═N), 150.1 (d, ²J_(CP1)=6 Hz, C₁ ¹), 150.5 (s, C₀ ¹).

Dendrimer 4-[G₃] (quantitative yield):

³¹P {¹H} NMR (d₆-DMSO): δ=6.9 (P₀), 61.9 (P_(1,2)), 68.1 (P₃).

¹H NMR (d₆-DMSO): δ=1.3 (br s, 288H, CH₂CH₃), 3.1 (s, 144H, ⁺N—CH₃),3.1–3.6 (m, 510H, CH₃—N—P_(1,2,3), CH₂), 5.5 (br s, 48H, N—H), 7.0–7.4(m, 84H, C₀ ²—H, C₁ ²—H, C₂ ²—H), 7.7–8.2 (m, 126H, CH═N, C₀ ³—H, C₁³—H, C₂ ³—H).

¹³C {¹H} NMR (d₆-DMSO): δ=7.7 (s, CH₂CH₃), 32.2 (d, ²J_(CP3)=9.4 Hz,CH₃—N—P₃), 33.5 (m, CH₃—N—P_(1,2)), 34.9 (s, CH₂—N—P₃), 47.3 (s,⁺N—CH₃), 56.4 (s, CH₂CH₃), 58.8 (d, ³J_(CP3)=4.8 Hz, CH₂—CH₂—N—P₃),121.0 (br s, C₀ ², C₁ ², C₂ ²), 128.5 (br s, C₀ ³, C₁ ³, C₂ ³), 132.3(s, C₀ ⁴, C₁ ⁴), 133.2 (s, C₂ ⁴), 137.1 (d, ³J_(CP3)=12.1 Hz, CH═N),141.0 (br s, CH═N), 150.1 (d, ²J_(CP2)=6.2 Hz, C₂ ¹), 150.7 (br s, C₀ ¹,C₁ ¹).

Dendrimer 4-[G₄] (quantitative yield):

³¹P {¹H} NMR (d₆-DMSO): δ=6.3 (P₀), 61.6 (P_(1,2,3)), 68.0 (P₄).

¹H NMR (d₆-DMSO): δ=1.3 (br s, 576H, CH₂CH₃), 3.1 (s, 288H, ⁺N—CH₃,3.1–3.6 (m, 1038H, CH₃—N—P_(1,2,3,4), CH₂), 5.5 (br s, 96H, N—H),7.1–8.5 (m, 450H, C₆H₄, CH═N).

¹³C {¹H} NMR (d₆-DMSO): δ=7.8 (s, CH₂CH₃), 32.2 (d, ²J_(CP4)=9.5 Hz,CH₃—N—P₄), 33.6 (d, ²J_(CP)=7.6 Hz, CH₃—N—P_(1,2,3)), 34.9 (s,CH₂—N—P₄), 47.3 (s, ⁺N—CH₃), 56.4 (s, CH₂CH₃), 58.7 (d, ³J_(CP4)=4.8 Hz,CH₂—CH₂—N—P₄), 121.2 (br s, C₀ ², C₁ ², C₂ ², C₃ ²), 128.2 (br s, C₀ ³,C₁ ³, C₂ ³, C₃ ³), 132.2 (s, C₀ ⁴, C₁ ⁴, C₂ ⁴), 133.2 (s, C₃ ⁴), 137.1(d, ³J_(CP4)=9.4 Hz, CH═N), 140.8 (br s, CH═N), 150.0 (d, ²J_(CP2)=6.2Hz, C₃ ¹), 150.7 (br s, C₀ ¹, C₁ ¹, C₂ ¹).

Dendrimer 4-[G₅] (quantitative yield):

³¹P {¹H} NMR (d₆-DMSO): δ=61.6 (P_(1,2,3,4)), 68.0 (P₅). ¹H NMR(d₆-DMSO): δ=1.3 (br s, 1152H, CH₂CH₃), 3.1 (s, 576H, ⁺N—CH₃), 3.1–3.6(m, 2094H, CH₃—N—P_(1,2,3,4,5), CH₂), 5.4 (m, 192H, N—H), 7.2–8.5 (m,930H, C₆H₄, CH═N). ¹³C {¹H} NMR (d₆-DMSO): δ=7.8 (s, CH₂CH₃), 32.3 (brs, CH₃—N—P₅), 33.5 (br s, CH₃—N—P_(1,2,3,4)), 34.9 (s, CH₂—N—P₅), 47.3(s, ⁺N—CH₃), 56.4 (s, CH₂CH₃), 58.8 (d, ³J_(CP)=5.0 Hz, CH₂—CH₂—N—P₅),121.3 (br s, C₀ ², C₁ ², C₂ ², C₃ ², C₄ ²), 128.2 (br s, C₀ ³, C₁ ³, C₂³, C₃ ³, C₄ ³), 132.2 (s, C₀ ⁴, C₁ ⁴, C₂ ⁴, C₃ ⁴), 133.2 (s, C₄ ⁴),137.2 (d, ³J_(CP4)=9.7 Hz, CH═N), 141.2 (br s, CH═N), 150.1 (d,²J_(CP2)=6.0 HZ, C₄ ¹), 150.7 (br s, C₀ ¹, C₁ ¹, C₂ ¹, C₃ ¹).

General Procedures for Synthesizing the Products of the 5-[G_(n)]Type

The resin with a high capacity for anion exchange, AG1-X8, was added toa suspension of 100 mg of methylated dendrimers 4-[G_(n)] (in the formof iodides) (n=1, 22 μmol; n=2, 10 μmol; n=3, 4.7 μmol; n=4, 21 μmol;n=5, 1.1 μmol) in distilled water (n=1, 25 ml; n=2, 23 ml; n=3, 22 ml;n=4, 21 ml; n=5, 20 ml), in the proportions indicated (n=1, 1.24 g; n=2,1.13 g; n=3, 1.06 g; n=4, 1.03 g; n=5, 1 g), and mixed gently for onehour. The paste obtained was washed with 20 ml of a pentane/ether (1/1,v/v) mixture so as to obtain a white powder of methylated dendrimers, inacetate form, named 5-[G_(n)]. 8 to 10% of the terminal branches aremodified during the counter-ion exchange with the resin and wereprovisionally attributed to a demethylated form since the NMR resultswere identical to those of the neutral dendrimers bearing tertiaryamines (3-[G_(n)]). These minor terminal branches were indicated with anasterisk in the NMR results present below and the yields correspond tothose of the total dendrimer.

Dendrimer 5-[G₁] (yield 90%):

³¹P {¹H} NMR (CD₃OD): δ=7.8 (P₀), 69.8 (P₁*), 70.3 (P₁).

¹H NMR (d₆-DMSO): δ=1.0 (t, ³J_(HH)=6.9 Hz, 6H, CH₂CH₃*), 1.3 (t,³J_(HH)=6.5 Hz, 66H, CH₂CH₃), 1.7 (s, 33H, CH₃COO⁻), 2.5 (m, 6H,CH₂*-N(CH₂*CH₃)₂), 3.1 (s, 33H, ⁺N—CH₃), 3.2 (d, ³J_(HP1)=8.3 Hz, 18H,CH₃—N—P₁), 3.3–3.7 (m, 90H, CH₂), 7.1 (d, ³J_(HH)=8.3 Hz, 12H, C₀ ²—H),7.6 (b, 12H, N—H), 7.8 (s, 6H, CH═N), 7.8 (d, ³J_(HH)=8.3 Hz, 12H, C₀³—H).

¹³C {¹H} NMR (d₆-DMSO): δ=7.6 (s, CH₂CH₃), 11.9 (s, CH₂C*H₃), 25.6 (s,CH₃COO⁻), 32.2 (d, ²J_(CP1)=9.5 Hz, CH₃—N—P₁), 38.8 (s, CH₂—N—P₁), 46.6(s, C*H₂CH₃), 47.0 (s, ⁺N—CH₃), 54.5 (s, C*H₂—CH₂—N—P₁), 56.2 (s,CH₂CH₃), 59.1 (d, ³J_(CP1)=5.3 Hz, CH₂—CH₂—N—P₁), 120.6 (s, C₀ ²), 127.9(s, C₀ ³), 133.7 (s, C₀ ⁴), 135.8 (d, ²J_(CP0)=10.8 Hz, CH═N), 149.8 (s,C₀ ¹), 173.7 (s, CH₃COO⁻).

UV-vis (H₂O): λ_(max) (ε, M⁻¹×cm⁻¹) 284 nm (1.2×10⁵).

Dendrimer 5-[G₂] (yield=95%):

³¹P {¹H} NMR (CD₃OD): δ=8.4 (P₀), 62.3 (P₁), 69.5 (P₂*), 70.1 (P₂).

¹H NMR (d₆-DMSO): δ=1.0 (br t, 12H, CH₂CH₃*), 1.3 (br s, 132H, CH₂CH₃),1.7 (s, 66H, CH₃COO⁻), 2.5 (br m, 12H, CH₂*-N(CH₂*CH₃)₂), 3.1 (s, 66H,⁺N—CH₃), 3.2–3.6 (m, 234H, CH₃—N—P_(1,2), CH₂) 7.1–7.3 (br m, 36H, C₀²—H, C₁ ²—H), 7.6 (br s, 24H, N—H), 7.7 (s, 12H, CH═N), 7.8–8.1 (br m,42H, CH═N, C₀ ³—H, C₁ ³—H).

¹³C {¹H} NMR (d₆-DMSO): δ=7.6 (s, CH₂CH₃), 11.9 (s, CH₂C*H₃), 25.6 (S,CH₃COO⁻), 32.1 (d, ²J_(CP2)=9.3 Hz, CH₃—N—P₂), 33.2 (d, ²J_(CP1)=13.4Hz, CH₃—N—P₁), 35.0 (s, CH₂—N—P₂), 46.6 (s, C*H₂CH₃), 47.0 (s, ⁺N—CH₃),54.5 (s, C*H₂—CH₂—N—P₂), 56.2 (s, CH₂CH₃), 59.1 (d, ³J_(CP2)=5.3 Hz,CH₂—CH₂—N—P₂), 121.3 (s, C₀ ², C₁ ²), 127.9 (s, C₁ ³), 128.5 (s, C₀ ³),132.3 (s, C₀ ⁴), 133.8 (s, C₁ ⁴), 135.7 (br s, CH═N), 149.8 (d,²J_(CP1)=6.5 Hz, C₁ ¹), 150.7 (s, C₀ ¹), 173.7 (s, CH₃COO⁻).

UV-vis (H₂O): λ_(max) (ε, M⁻¹×cm⁻¹) 284 nm (4.1×10⁵).

Dendrimer 5-[G₃] (yield=95%):

³¹P {¹H} NMR (CD₃OD): δ=8.4 (P₀), 62.3 (P_(1,2)), 69.5 (P₃*), 70.1 (P₃).

¹H NMR (d₆-DMSO): δ=1.0 (br t, 30H, CH₂CH₃*), 1.3 (br s, 258H, CH₂CH₃),1.7 (s, 129H, CH₃COO⁻), 2.5 (br m, 30H, CH₂*-N(CH₂*CH₃)₂), 3.1 (s, 129H,⁺N—CH₃), 3.1–3.6 (m, 480H, CH₃—N—P_(1,2,3), CH₂) 7.1–7.5 (br m, 108H, C₀²—H, C₁ ²—H, C₂ ²—H, N—H), 7.5–8.1 (br m, 126H, CH═N, C₀ ³—H, C₁ ³—H, C₂³—H).

¹³C {¹H} NMR (d₆-DMSO): δ=7.6 (s, CH₂CH₃), 11.9 (s, CH₂C*H₃), 25.2 (s,CH₃COO⁻), 32.1 (d, ²J_(CP3)=9.0 Hz, CH₃—N—P₃), 33.2 (br m,CH₃—N—P_(1,2)), 35.0 (s, CH₂—N—P₃), 46.6 (s, C*H₂CH₃), 47.1 (s, ⁺N—CH₃),54.5 (s, C*H₂—CH₂—N—P₃), 56.3 (s, CH₂CH₃), 59.1 (d, ³J_(CP3)=5.7 Hz,CH₂—CH₂—N—P₃), 121.3 (br s, C₀ ², C₁ ², C₂ ²), 128.0 (s, C₂ ³), 128.5(S, C₀ ³, C₁ ³), 132.4 (s, C₀ ⁴, C₁ ⁴), 133.8 (s, C₂ ⁴), 136.3 (br d, C₂⁴—CH═N), 141.0 (br m, CH═N), 150.1 (d, ²J_(CP2)=6.3 Hz, C₂ ¹), 151.0 (d,²J_(CP)=7.1 Hz, C₀ ¹, C₁ ¹), 173.7 (s, CH₃COO⁻) .

UV-vis (H₂O): λ_(max) (ε, M⁻¹×cm⁻¹) 286 nm (7.4×10⁵).

Dendrimer 5-[G₄] (yield=95%):

³¹P {¹H} NMR (CD₃OD): δ=8.2 (P₀), 62.2 (P_(1,2,3)), 69.9 (P₄).

¹H NMR (d₆-DMSO): δ=1.0 (br m, 60H, CH₂CH₃*), 1.3 (br s, 516H, CH₂CH₃),1.7 (s, 258H, CH₃COO⁻), 2.5 (m, 60H, CH₂*-N(CH₂*CH₃)₂), 3.1 (s, 258H,⁺N—CH₃), 3.1–3.6 (m, 978H, CH₃—N—P_(1,2,3,4), CH₂) 7.0–8.2 (m, 546H,C₆H₄, CH═N, N—H).

¹³C {¹H} NMR (d₆-DMSO): δ=7.6 (s, CH₂CH₃), 11.9 (s, CH₂C*H₃), 24.7 (s,CH₃COO⁻), 32.1 (d, ²J_(CP4)=9.6 Hz, CH₃—N—P₄), 33.2 (d, ²J_(CP)=11.2 Hz,CH₃—N—P_(1,2,3)), 35.0 (s, CH₂—N—P₄), 46.6 (s, C*H₂CH₃), 47.0 (s,⁺N—CH₃), 54.5 (d, ³J_(CP4)=3.9 Hz, C*H₂—CH₂—N—P₄), 56.3 (s, CH₂CH₃),59.2 (d, ³J_(CP4)=5.7 Hz, CH₂—CH₂—N—P₄), 121.2 (s, C₃ ²), 121.6 (s, C₀², C₁ ², C₂ ²), 127.9 (s, C₃ ³), 128.5 (s, C₀ ³, C₁ ³, C₂ ³), 132.3 (s,C₀ ⁴, C₁ ⁴, C₂ ⁴), 133.7 (s, C₃ ⁴), 135.8 (br s, C₃ ⁴—CN═N), 141.0 (brs, CH═N), 149.9 (d, ²J_(CP3)=6.3 Hz, C₃ ¹), 151.0 (br m, CO₀ ¹, C₁ ¹, C₂¹), 173.7 (s, CH₃COO⁻).

UV-vis (H₂O): λ_(max) (ε, M⁻¹×cm⁻¹) 288 nm (1.6×10⁶).

Dendrimer 5-[G₅] (yield=95%):

³¹P {¹H} NMR (d₆-DMSO): δ=62.2 (P_(1,2,3,4)), 69.9 (P₅).

¹H NMR (d₆-DMSO): δ=1.0 (m, 114H, CH₂CH₃*), 1.3 (m, 1038H, CH₂CH₃), 1.7(s, 519H, CH₃COO⁻), 2.5 (m, 114H, CH₂*-N(CH₂*CH₃)₂), 3.1 (s, 519H,⁺N—CH₃), 3.1–3.6 (m, 1980H, CH₃—N—P_(1,2,3,4,5), CH₂), 7.0–8.2 (m,1122H, C₆H₄, CH═N, N—H).

¹³C {¹H} NMR (d₆-DMSO): δ=7.6 (s, CH₂CH₃), 11.9 (s, CH₂C*H₃), 24.7 (s,CH₃COO⁻), 32.1 (d, ²J_(CP5)=9.6 Hz, CH₃—N—P₅), 33.2 (d, ²J_(CP)=11.2 Hz,CH₃—N—P_(1,2,3,4)), 35.0 (s, CH₂—N—P₅), 46.6 (s, C*H₂CH₃), 47.0 (s,⁺N—CH₃), 54.5 (d, ³J_(CP5)=3.9 Hz, C*H₂—CH₂—N—P₅), 56.3 (s, CH₂CH₃),59.2 (d, ³J_(CP5)=5.7 Hz, CH₂—CH₂—N—P₅), 121.2 (s, C₀ ², C₁ ², C₂ ², C₃², C₄ ²), 127.9 (s, C₄ ³), 128.5 (s, C₀ ³, C₁ ³, C₂ ³, C₃ ³), 132.3 (s,C₀ ⁴, C₁ ⁴, C₂ ⁴, C₃ ⁴), 133.7 (s, C₄ ⁴), 135.9 (br s, C₄ ⁴—CH═N), 141.0(br s, CH═N), 149.9 (d, ²J_(CP3)=6.5 Hz, C₄ ¹), 151.0 (br m, C₀ ¹, C₁ ¹,C₂ ¹, C₃ ¹), 173.7 (s, CH₃COO⁻).

UV-vis (H₂O): λ_(max) (ε, M⁻¹×cm⁻¹) 284 nm (3.7×10⁶).

EXAMPLE 2 Transfection Experiments

The results obtained with these various polycationicphosphorus-containing dendrimers always showed optimum efficiency withan N/P charge ratio of between 5 (FIG. 4) and 10 (FIG. 5) (N/Pratio=number of terminal nitrogen atoms of the dendrimer per phosphateof the DNA). Thus, it was decided, in an arbitrary manner, to comparethe transfection efficiency of various generations ofphosphorus-containing dendrimers (P-dendrimers) with that of the linearPEI ExGen 500, having 5 to 10 amine equivalents per nucleotide.

With 5 positive-charge equivalents per phosphate of the DNA, the 5different generations of protonated dendrimers of the 2-[G_(n)] type,tested in the form of hydrochlorides, made it possible to obtainsignificant expression of the transgene. An increase of 10⁵ to almost10⁹ relative light units (RLU)/mg of protein was observed. Thetransfection efficiency increases with the size of the dendrimer(generation number) but reaches a plateau from the third generation,with values of between 10⁸ ad 10⁹ RLU (FIG. 4). Consequently, 2-[G₄] waschosen to study in greater detail the transfection efficiency of thesenovel cationic phosphorus-containing dendrimers, the structure of whichis given in FIG. 2.

It should be noted that the transfections performed in the presence ofserum produce less toxicity. As a result, a higher level of expressionis observed for the transfections performed in the presence of serumcompared to those performed without serum (FIGS. 4A and 4B, right andleft panels, respectively).

Without trying to further optimize their transfection conditions, thedendrimers of the third to the fifth generation prove to be as efficientas the linear PEI used under optimal conditions.

On the other hand, the methylated forms 5-[G_(n)] prove to be rathertoxic and relatively inefficient for transfecting nucleic acids intoeukaryotic cells (FIGS. 5A and 5B). This phenomenon may be explained bya stable positive charge density which may disturb the cell membrane andcause cell death. It is not possible to decrease the charge density ofthe methylated forms without degrading the dendrimer; on the other hand,the charge density of the chloride derivatives (2-[G_(n)]) may bemodulated by microenvironmental modifications of the pH when theyapproach the cell membrane. In addition, the possibility of modulatingthe charge density of the chloride derivatives of the dendrimers mayplay a key role in the release of the luciferase gene from the endosome.These dendrimers perhaps behave like proton reservoirs in the cellularcompartments, their charge density being controlled, firstly, byATPase-coupled proton pumps and, secondly, by intracellularmodifications of the chloride concentration. The possibility ofdecreasing the cationic charge density of these phosphorus-containingdendrimers, inside or outside the cells, would be an advantage forcarrying out experiments in vivo, as previously mentioned (26).

Chemical Products

The linear 22 kDa PEI (ExGen 500) was supplied by Euromedex(Souffelweyersheim, France).

Cell Lines and Culture

The NIH3T murine fibroblasts come from the ATCC (Rockville, Mass., USA)and are cultured in Dulbecco modified Eagle medium (DMEM). The culturemedia are supplemented with 10% of fetal calf serum (D. Dutscher,Brunath, France), 2 mM of L-glutamine (Gibco-BRL), 100 units/ml ofpenicillin (Gibco-BRL) and 100 μg/ml of streptomycin (Gibco-BRL). Thecells are maintained at 37° C., in a humid atmosphere containing 5% ofCO₂. When the cells reach 80% confluency, they are detached with asaline solution of trypsin-EDTA (Gibco-BRL), diluted ten-fold andcultured in a new flask.

Plasmids

PCMV-luc, which encodes Photinus pyralis luciferase under the control ofthe promoter/enchancer sequences of the cytomegalovirus were kindlyprovided by Dr. M. Scarpa (CRIBI, Padoua, Italy). The plasmids werepurified on Qiagen columns (Rockford, USA), from the transformed E. colistrain XL1.

Transfection of Cells

The adherence cells were seeded into 24-well plates (Costar, D.Dutscher, France) the day before transfection, so as to reach 60 to 70%confluency on the day of transfection. All the experiments were carriedout in triplicate. The cells were rinsed before transfection and 1 ml ofmedium with or without serum was added to each well. 2 μg of plasmid(1.5 mg/ml solution in a 10 mM Tris-1 mM EDTA buffer, pH 7.4) werediluted in 50 μl of 0.15M NaCl.

The N/P (nitrogen/phosphate) ratio corresponds to the amount of polymernecessary to have one amine residue (43 Da=average molecular weight(M_(w)) for the PEI; for the dendrimers, the nitrogen molarity of theamine residues was calculated by dividing the M_(w) by the number ofcharges for each generation) per nucleic acid phosphate (M_(w)330) (12).The required amount of linear PEI (ExGen500) and of dendrimers (fromstock solutions of PEI and of dendrimers corresponding to 10 mM ofnitrogen, in amine form, in sterile MilliQ water) was diluted in 50 μlof 0.15M NaCl, vortexed gently and centrifuged. After 15 min, thecationic vector was added, in a single step, to the plasmid solution[and not in the reverse order (12)] and the mixture was then vortexedand centrifuged. The amounts and volumes indicated above correspond toone well and were, in fact, multiplied by three and distributed intothree wells. After 10 min, the mixture was added to the cells and thesupernatant was distributed homogeneously by slight horizontal manualrotation. Immediately afterwards, the culture plate was centrifuged(Sigma 3K10, Bioblok, France) for 5 min at 1 500 rpm (280 g). Afterincubation for 2 to 3 hours, 110 μl of fetal calf serum were added tothe wells without serum. The cells were cultured for 24 h and theexpression of the reporter gene was then tested.

Luciferase Measurement

The expression of the luciferase gene was measured by luminescence. Theculture medium was removed and the cell lysate was harvested afterincubation for 30 min at room temperature in the 1× lysis buffer(Promega, USA). The lysate was gently vortexed and centrifuged at 140000 rpm (17 530 g), for 5 min at 4° C. 20 μl of lysate were diluted in100 μl of luciferase reaction buffer (Promega, USA) and the luminescencewas measured for 10 sec (Mediators PhL, Vienna, Austria). Results wereexpressed as units of luminescence per mg of protein (measurement usingthe BCA test, Pierce).

EXAMPLE 3 Percentages of Viability of the Cells Transfected inAccordance with Example 2

Dendrimers-N⁺Et₂H, Cl⁻ Dendrimers-N⁺Et₂Me, CH₃CO₂ ⁻ 5 10 5 10equivalents equivalents equivalents equivalents G₁ 95 105 G₁ 90 88 G₂ 9183 G₂ 91 101 G₃ 83 90 G₃ 87 99 G₄ 94 91 G₄ 99 95 G₅ 90 80 G₅ 108 92Linear PEI: 5 equivalents: 69; 10 equivalents: 84 Crosslinked PEI: 5equivalents: 102; 10 equivalents: 91

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As emerges from the above, the invention is in no way limited to itsmethods of implementation, preparation and application which has justbeen described more explicitly; on the contrary, it encompasses all thevariants thereof which may occur to a person skilled in the art, withoutdeparting from the context or the scope of the present invention.

1. A polycationic phosphorus-containing dendrimer, which consists: of acentral layer in the form of a central core P₀ comprising 2 to 8functionalized groups, of n intermediate layers, which may be identicalor different, each one of said intermediate layers consisting of unitsP₁ corresponding to formula II:

in which; L is an oxygen, phosphorus or sulfur atom, M represents one ofthe following groups: an aromatic group di-, tri- or tetra-substitutedwith alkyl, alkoxy or unsaturated C₁–C₁₂ olefinic, azoic or acetylenicgroups, unsubstituted or substituted with at least one phosphorous,oxygen, nitrogen, sulfur or halogen atom, or an alkyl or alkoxy groupcomprising several substituents as defined when M is an aromatic group,R₁ and R₂, which may be identical or different, represent a hydrogenatom or one of the following groups: alkyl, alkoxy, or aryl,unsubstituted or substituted with at least one phosphorous, oxygen,nitrogen, sulfur or halogen atom, n is an integer between 1 and 11, E isselected from the group consisting of an oxygen atom, a sulfur atom, anitrogen atom and a nitrogen atom linked to an alkyl, alkoxy or arylgroup, an outer layer consisting of units P2, which may be identical ordifferent, corresponding to formula III:

in which: R₅ represents a hydrogen atom or one of the following groups:alkyl, alkoxy, or aryl, unsubstituted or substituted with at least onephosphorus, oxygen, nitrogen, sulfur or halogen atom, W represents oneof the following groups: alkyl, alkoxy, or aryl, unsubstituted orsubstituted with at least one phosphorus, oxygen, nitrogen, sulfur orhalogen atom, R₃ and R₄, which may be identical or different, representa C₁–C₅ alkyl group, X represents a hydrogen atom or a C₁–C₅ alkyl groupor is absent, and Z represents a halide ion, an alkylCOO⁻ group or anyother anionic group comprising carbon, oxygen, sulfur, nitrogen,phosphorus or halogen atoms, or is absent.
 2. The dendrimer as claimedin claim 1, wherein the central core P₀ is selected from the groupconsisting of the group of general formula Ia:

and the group of general formula Ib:


3. The dendrimer as claimed in claim 1 or claim 2, wherein saiddendrimer has terminal units of formula III, in which X represents ahydrogen atom, R₅ is a hydrogen atom, W represents a group (CH₂)₂, R₃and R₄ are identical and represent ethyl groups, and Z is a chlorideion.
 4. The dendrimer as claimed in claim 1 or claim 2, wherein saiddendrimer has terminal units of formula III, in which X represents amethyl group, R₅ is a hydrogen atom, W represents a group (CH₂)₂, R₃ andR₄ are identical and represent ethyl groups, and Z is a group CH₃COO⁻.5. A composition capable of acting as an agent for transfecting anucleic acid sequence into a eukaryotic cell, said compositioncomprising a nucleic acid and a polycationic phosphorus-containingdendrimer as claimed in claim 1, coupled to said nucleic acid.
 6. Thecomposition as claimed in claim 5, said composition further comprisingat least one pharmaceutically acceptable vehicle.
 7. The composition asclaimed in claim 5 or claim 6, wherein the N/P ratio, in which Ncorresponds to the number of terminal nitrogen atoms of the dendrimerper phosphate (P) of said nucleic acid, is between 5 and
 10. 8. Thecomposition as claimed in claim 5, said composition further comprisingan agent for permeabilizing the membrane, capable of transporting saidnucleic acid across the cytoplasmic or endosomal membranes of saideukaryotic cell.
 9. The composition as claimed in claim 5, wherein saidpolycationic phosphorus-containing dendrimer is associated noncovalentlywith said nucleic acid.